Myofibrogranuloma (MFG) is a degenerative disease of skeletal muscle recognized only in walleye (Stizostedion vitreum). MFG is a unique form of skeletal degeneration. The disease is characterized by profound alterations of the trunk musculature produced by extensive hypertrophy of the muscle fibers. Two degenerative processes are involved. The first and most pronounced lesion consists of coagulation necrosis of muscle fibers accompanied by an inflammatory response and formation of granulomas (muscle tumors). The second is non-inflammatory and characterized by local areas of acute myolysis. Opaque, yellowish brown muscle with a sandy texture is seen in fish with advanced forms of the myopathy. Fishermen will usually discard fish with advanced MFG because the lateral trunk muscles (fillet) especially beneath the skin becomes sandy, discolored and somewhat jelly-like.
MFG has been recognized by disease specialists to occur most frequently in older fish. Economon suggests MFG to be present in 1 of 10,000 walleye 4.5 years or older. Economon, P. P., "Myofibrogranuloma, a Muscular Dystrophy-Like Anomaly of Walleye (Stizostedion vitreum), Minnesota Dep. Nat. Resour. Fish Wildl. Spec. Publ., 113: 1-11 (1975). The mean total length at capture for walleye five years of age or older is generally greater than or equal to 500 mm.
The general nature of the muscle degeneration suggests MFG is of a hereditary nature. It has been suggested that MFG in walleye is apparently not sex-linked and that MFG represents a unique myopathy. Kelly, et al., "Chemical analysis of muscle from walleye (Stizostedion vitreum vitreum) with myofibrogranuloma, a chronic myopathy," Can. J. Fish. Aquat. Sci. 44: 1425-1431 (1987). Further, it has been demonstrated that the disease is most likely not infectious. Holloway, et al., "A myopathy in North Dakota walleye, Stizostedion vitreum," J. Fish Dis. 5: 527-530 (1982) Thus, due to MFG's probable genetic transmission, shipment of potentially diseased eggs is a concern of fishery biologists.
In addition, many lakes and reservoirs have increasingly used stocking as a management tool. For instance, at the Merritt Reservoir in north central Nebraska, the walleye population is maintained almost exclusively by stocking (Joel Klammer, Nebraska Game and Parks Commission, personal communication, Lincoln, Nebr. 68503-0370). Further, in 1990, over 6.2 million walleye fingerlings were stocked into Lake Sakakawea, North Dakota, to enhance the population. In lower Lake Oahe 221,250(1986), 299,500 (1987), 476,868 (1988) and 449,256 (1989) fingerling walleye were stocked. Fielder, D. G., "Evaluation of stocking walleye fry and fingerlings and factors affecting their success in lower Lake Oahe, South Dakota," North Am. J. Fish. Mgmt. 12: 336-345 (1992a); Fielder, D. G., "Relationship between walleye fingerling stocking density and recruitment in lower Lake Oahe, South Dakota," North Am. J. Fish. Mgmt. 12: 346-352 (1992b). Moreover, multiple years of almost complete year class failure and angler demand on the reservoirs have led to the increased stocking of walleye to enhance and even establish year classes.
MFG is of concern primarily in the Midwest, particularly in North and South Dakota, Minnesota, and Nebraska. Roe shipped from those states is sent throughout the country, wherever walleye is stocked for sport fishing. Therefore, a field test for this disease would be useful to remove diseased animals from the population and minimize the risk of spread via roe.
Present methods for detecting MFG can not be accomplished without filleting the fish since walleye with MFG generally show no external symptoms or abnormal behavior. Kelly et al. chemically analyzed 15 cases of MFG in adult walleye from 1975 to 1985 from seven lakes in southern Manitoba and northwestern Ontario and from Lake Sakakawea, North Dakota. Kelly, R. K., et al., "Chemical Analysis of Muscle From Walleye with Myofibrogranuloma, a Chronic Myopathy," Can. J. Fish. Aquat. Sci. 45: 1425-1431 (1987). The inorganic analysis of representative samples revealed elevations or depressions of ions which were consistent with those of extensively damaged muscle membrane systems. One of the greatest differences was seen in calcium levels which averaged 55 times normal in muscle lesions where excessive calcium deposition was demonstrable by histochemical staining with nuclear fast red. Id.
X-ray analysis has been utilized to show calcium deposits in the human kidney by Davidson, who stated that technicians are able to visualize various organs and systems radiographically because of differential absorption of X-rays, owing to differences in density of materials being examined. Davidson, A. J., "Excretory Urography", Computed Tomography, Ultrasound and X-Ray: An Integrated Approach, Masson Publishing U.S.A., Inc., New York, pp. 11-20 (1979). Thus, applicants hypothesized that muscles affected by MFG would have a higher density than normal muscle tissue, owing to the influx of calcium and subsequent calcification in advanced cases which could be detected with X-rays. The disadvantages of using X-rays, however, is that X-rays can be harmful to fish and would make subsequent genetic studies questionable. Therefore, there is a need in the art for a means of diagnosing MFG without filleting the fish or using X-rays.
Vascular biochemistry seemed the most likely choice since human muscular dystrophy is diagnosed with blood chemistry values. Many specific diseases, whether caused by bacteria, viruses or metabolic dysfunctions, are characterized by specific changes in the composition of the blood. Shell, E. W., "Chemical Composition of Blood of Smallmouth Bass," Res. Rep. U.S. Fish Wildl., Serv. No. 57, U.S. Government Printing Office, Washington D.C. (1961). Diagnosis depends on the comparison of blood composition values of the animal with the suspected disease with blood composition of healthy animals of the same species.
Oser states that the normal range for calcium is narrow and small variations indicate pathology such as bone abnormalities and muscle tumors. Oser, B. L., Hawk's Physiological Chemistry, McGrawHill Book Company, New York, New York (1965). If calcium is regulated in the walleye, fish exhibiting MFG should have significantly different serum calcium levels than healthy walleye due to the 55-fold increase in muscle calcium associated with MFG shown by Kelly et al. in 1987. It was hypothesized that MFG-diseased walleye would exhibit relatively higher serum calcium concentrations than healthy walleye.
In 1993, it was discovered that there was a significant difference between mean serum calcium levels in spawning male and spawning female walleye. Serum calcium is higher in females due to the influence of estrogen which stimulates synthesis of yoke protein and an increase in protein bound plasma calcium. It was further found that the mean serum calcium levels of spawning male walleye was significantly different from the mean serum calcium levels of MFG-positive spawning male walleye. Similarly, there was a significant difference found between mean serum calcium levels of spawning female walleye and mean serum calcium levels of MFG-positive spawning female walleye. These elevations suggested that calcium was regulated in the walleye and that MFG was related to serum calcium levels. The calcium serum level test for MFG was not definitive, however, since there was an overlap of ranges between the healthy and MFG-positive spawning females and males. Therefore, it was determined that serum calcium levels, in and of themselves, could not be used to accurately diagnose MFG in walleye.
According to the invention, creatinine is another vascular parameter which potentially may be used to assess muscle degeneration. Phosphocreatine in muscle spontaneously cycles to creatinine. The quantity of end product, creatinine, is proportional to muscle mass and thus may be utilized in evaluating muscle mass. It was hypothesized that creatinine concentration in walleye serum would reflect these muscle mass-phosphocreatine/creatinine relationships. It was further hypothesized that since muscle mass decreases in MFG-afflicted walleye, albeit up to 20% in the most severe cases, that phosphocreatine may also decrease. A decrease in phosphocreatine means a reduction in the spontaneous cyclization to creatinine. If there is a reduction in creatinine levels in MFG-positive walleye, the values in the blood serum might reflect this.
Therefore, a primary objective of the present invention is to utilize a method for diagnosing MFG in walleye which is accurate and is not harmful to the fish. This invention has as its primary objective the fulfillment of this need.
Another objective of the present invention is to provide a method of diagnosing MFG in walleye which is more accurate than previously available methods.
Yet another objective of the present invention is to provide a method for diagnosing MFG which is easy to perform and cost effective.
The method and means of accomplishing these and further objectives of the invention will become apparent from the detailed description which follows hereinafter.